The present invention relates to the field of cationic lipid compounds and their uses including the delivery of macromolecules into cells.
None of the following discussion of the background of the invention is admitted to be prior art to the invention.
Lipid aggregates, such as liposomes, have been previously reported to be useful as agents for the delivery of macromolecules (such as DNA, RNA, oligonucleotides, proteins, and pharmaceutical compounds) into cells. In particular, lipid aggregates, which include charged as well as uncharged lipids, have been described as being especially effective for delivering polyanionic molecules to cells. The reported effectiveness of cationic lipids may result from charge interactions with cells which are said to bear a net negative charge. It has also been postulated that the net positive charge on the cationic lipid aggregates may enable them to bind polyanions, such as nucleic acids. For examples, lipid aggregates containing DNA have been reported to be effective agents for efficient transfection of cells.
The structure of a lipid aggregate depends on factors which include composition of the lipid and the method of forming the aggregate. Lipid aggregates include, for example, liposomes, unilamellar vesicles, multilamellar vesicles, micelles and the like, and may have particle sizes in the nanometer to micrometer range. Various methods of making lipid aggregates have been reported in the art. One type of lipid aggregate includes phospholipid containing liposomes. An important drawback to the use of this type of aggregate as a cell delivery vehicle is that the liposome has a negative charge that reduces the efficiency of binding to a negatively charged cell surface. It has been reported that positively charged liposomes that are able to bind DNA may be formed by combining cationic lipid compounds with phospholipids. These liposomes then be utilized to transfer DNA into target cells. (See, e.g. Felgner et al., Proc. Nat. Acad. Sci. 84:7413-7417, 1987; Eppstein et al. U.S. Pat. No. 4,897,355; Felgner et al. U.S. Pat. No. 5,264,618; and Gebeyehu et al. U.S. Pat. No. 5,334,761).
Known cationic lipids include N[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium chloride (xe2x80x9cDOTMAxe2x80x9d). Combinations of DOTMA with dioleoylphosphatidylethanolamine (xe2x80x9cDOPExe2x80x9d) have been commercially available. Formulation of DOTMA, either by itself or in 1:1 combination with DOPE, into liposomes by conventional techniques has been reported. However, compositions comprising DOTMA have been reported to show some toxicity to cells.
Another commercially available cationic lipid, 1,2-bis (oleoyloxy)-3,3-(trimethylammonia)propane (xe2x80x9cDOTAPxe2x80x9d) differs from DOTMA in structure in that the oleoyl moieties are linked by ester, rather than ether, linkages to the propylamine. See Figure. However, DOTAP is reported to be more readily degraded by target cells. Other cationic lipids which represent structural modifications of DOTMA and DOTAP have also been reported.
Other reported cationic lipid compounds include those in which carboxyspermine has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (xe2x80x9cDOGSxe2x80x9d) and dipalmitoyl-phosphatidylethanolamine 5-carboxyspermyl-amide (xe2x80x9cDPPESxe2x80x9d) (See, e.g. Behr et al., U.S. Pat. No. 5,171,678).
Another reported cationic lipid composition is a cationic cholesterol derivative (xe2x80x9cDC-Cholxe2x80x9d) which has been formulated into liposomes in combination with DOPE. (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). For certain cell lines, these liposomes were said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions.
Lipopolylysine, made by conjugating polylysine to DOPE has been reported to be effective for transfection in the presence of serum. (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991).
However, of the cationic lipids which have been proposed for use in delivering macromolecules to cells, no particular cationic lipid has been reported to work well with a wide variety of cell types. Since cell types differ from one another in membrane composition, different cationic lipid compositions and different types of lipid aggregates may be effective for different cell types, either due to their ability to contact and fuse with target cell membranes directly or due to different interactions with intracellular membranes or the intracellular environment. For these and other reasons, design of effective cationic lipids has largely been empirical. In addition to content and transfer, other factors believed important include, for example, ability to form lipid aggregates suited to the intended purpose, toxicity of the composition to the target cell, stability as a carrier for the macromolecule to be delivered, and function in an in vivo environment. Thus, there remains a need for improved cationic lipids which are capable of delivering macromolecules to a wide variety cell types with greater effeciency.
In one aspect of the present invention novel carbamate-based cationic lipids having the structure: 
or a salt, or solvate, or enantiomers thereof are provided wherein; (a) R1 is a lipophilic moiety; (b) R2 is a positively charged moiety; (c) n is an integer from 1 to 8; (d) Xxe2x88x92 is an anion or polyanion; and (e) m is an integer from 0 to a number equivalent to the positive charge(s) present on the lipid.
In one embodiment R1 may be selected from a variety of lipophilic moieties including a straight chain alkyl of 1 to about 24 carbon atoms, a straight chain alkenyl of 2 to about 24 carbon atoms, a symmetrical branched alkyl or alkenyl of about 10 to about 50 carbon atoms (preferably 25-40), an unsymmetrical branched alkyl or alkenyl of about 10 to about 50 carbon atoms, a steroidyl moiety, a glyceryl derivative or CH(R3R4), wherein R3 and R4 are independently a straight chain alkyl moiety of about 10 to about 30 carbon atoms, or a branched alkyl moiety of about 10 to about 30 carbon atoms.
Preferably when R1 is a steroidyl moiety it is a cholesteryl moiety or a non-cholesteryl moiety.
In a prefered embodiment R1 is 3-(1,2-diacyl)propane 1,2-diol moiety or a 3-(1,2-dialkyl)propane 1,2-diol moiety. In particular when R1 is a 3-(1,2-diacyl)propane 1,2-diol moiety itmis preferable that the diacyl group be alkanoic acid of about 10 to about 30 carbon atoms or an alkenoic acid of about 10 to about 30 carbon atoms. When R1 is 3-(1,2-dialkyl)propane 1,2-diol moiety it is preferable that the alkyl moieties be an alkyl group of about 10 to about 30 carbon atoms or an alkenyl group of about 10 to about 30 carbon atoms.
When R1 is a glyceryl derivative it is prefered that it be a 3-O-1,2-diacylglyceryl moiety or a 3-O-1,2-dialkylglyceryl moiety. In particular, when R1 is a 3-O-1,2-diacylglyceryl moiety it is preferable that the diacyl group be an alkanoic acid of about 10 to about 30 carbon atoms or an alkenoic acid of about 10 to about 30 carbon atoms.
It is particularly prefered that when a moiety contains an alkanoic acid that the acid be stearic and when a moiety contains an alkenoic acid that the acid be palmitoic acid or oleic acid.
In another prefered R1 is 18-pentatriacontane, 3-(3xcex2)-cholest-5-ene, or 3-(1,2 distearyl)propane 1,2-diol.
In another embodiment R2 may be selected from a variety of positively charged moieties including an amino acid residue having a positively charged group on the side chain, an alkylamine moiety of about 3 to about 10 carbon atoms, a fluoroalkylamine moiety, or a perfluoroalkylamine moiety of 1 to about 6 carbon atoms, an arylamine moiety or an aralkylamine moiety of 5 to about 10 carbon atoms, a guanidinium moiety, an enamine moiety, an aromatic or non-aromatic cyclic amine moiety of 3 to about 9 carbon atoms, an amidine moiety, an isothiourea moiety, a heterocyclic amine moiety, or a heterocyclic moiety or an alkyl moiety of 1 to about 6 carbon atoms substituted with a substituent selected from the group consisting of NH2, C(xe2x95x90O)NH2, NHR6, C(xe2x95x90O)NHR6, NHR6R7, or C(xe2x95x90O)NHR6R7, wherein R6 and R7 are independently selected from an alkyl moiety of 1 to about 24 carbon atoms, an alkenyl moiety of 2 to about 24 carbon atoms, an aryl moiety of about 5 to about 20 carbon atoms, and an aralkyl moiety of about 6 to about 25 carbon atoms.
Preferably when R2is an amino acid residue it is a lysine, arginine, histidine, ornithine, or an amino acid analog. In particular, it is prefered that the amino acid residue be 3-carboxyspermidine, 5-carboxyspermidine, 6-carboxyspermine or monoalkyl, dialkyl or peralkyl substituted derivatives which are substituted on one or more amine nitrogens with an alkyl group of 1 to about 6 carbon atoms.
It is further prefered that n be an integer from 2 to 6, or more preferably from 2 to 4 and that Xxe2x88x92 be a pharmaceutically acceptable anion or polyanion.
In a particularly prefered embodiment the carbamate-based cationic lipid has the structure 
In another aspect of the present invention, compositions comprising a polyanionic macromolecule and any of the lipids described above are provided. In particular, the macromolecule may be a variety of polyanionic macromolecules including an expression vector capable of expressing a polypeptide in a cell, In a prefered the polyanionic macromolecule is an oligomucleotide or an oligomer and most preferably DNA.
In still another aspect of the present invention methods for delivering a polyanionic macromolecule into a cell by contacting any of the compositions above with the cell are provided. In particular, a method is provided to interfere with the expression of a protein in a cell by contacting any of the compositions described above with a cell wherein the composition comprises an oligomer having a base sequence that is substantially complimentary to an RNA sequence in the cell that encodes the protein.
The present invention further provides a kit for delivering a polyanionic macromolecule into a cell comprising any of the compositions described above.
Prior to setting forth the remaining description of the invention, it may be helpful to an understanding thereof to first set forth definitions of certain terms that will be used herein after. These terms will have the following meanings unless explicitly stated otherwise:
xe2x80x9cLipophilic Moietyxe2x80x9d refers to a moiety which demonstrates one or more of the following characteristics:
In particular, lipophilic moieties having an octanol/water partition coefficinet of 0.5 or lower are preferable, where octanol/water partition coefficient is measured by the concentration in water divided by concentration in octanol.
xe2x80x9cPositively Charged Moietyxe2x80x9d refers to xe2x80x9cpositively charged moiety and negatively charged moietyxe2x80x9d means a moiety, independent of the cationic lipid for which it is a substituent, having a net positive or negative charge within the pH range of 2 to 12. The net charge of the cationic lipid is the summation of all charged moieties occuring on the lipid, such that the net charge may be positive, neutral or negative.
The term xe2x80x9calkylxe2x80x9d refers to saturated aliphatic groups including straight-chain, branched-chain and cyclic groups. Suitable alkyl groups include, but are not limited to, cycloalkyl groups such as cyclohexyl and cyclohexylmethyl. xe2x80x9cLower alkylxe2x80x9d refers to alkyl groups of 1 to 6 carbon atoms. Fluoroalkyl or perfluoroalkyl refers to singly, partially, or fully fluorinated alkyl groups.
The term xe2x80x9calkenylxe2x80x9d refers to an unsaturated aliphatic group having at least one double bond.
The term xe2x80x9carylaminexe2x80x9d refers to aromatic groups that have at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which are substituted with an amine.
The term xe2x80x9caralkylaminexe2x80x9d refers to an alkylamine group substituted with an aryl group. Suitable aralkyl groups include benzyl, picolyl, and the like, all of which may be optionally substituted.
The term xe2x80x9cheterocyclic aminexe2x80x9d refers to groups having from 1 to 3 heteroatoms as ring atoms in the aromatic ring and the remainder of the ring atoms are carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen, and suitable heterocyclic aryls include furanyl, thienyl, pyridyl, pyrrolyl, pyrimidyl, pyrazinyl, imidazolyl, and the like.
The term xe2x80x9coligonucleosidexe2x80x9d or xe2x80x9coligomerxe2x80x9d refers to a chain of nucleosides that are linked by internucleoside linkages that is generally from about 4 to about 100 nucleosides in length, but which may be greater than about 100 nucleosides in length. They are usually synthesized from nucleoside monomers, but may also be obtained by enzymatic means. Thus, the term xe2x80x9coligomerxe2x80x9d refers to a chain of oligonucleosides that have internucleosidyl linkages linking the nucleoside monomers and, thus, includes oligonucleotides, nonionic oligonucleoside alkyl- and aryl-carbamate analogs, alkyl- and aryl-carbamothioate, analogs of oligonucleotides, carbamate analogs of oligonucleotides, neutral carbamate ester oligonucleoside analogs, other oligonucleoside analogs and modified oligonucleosides, and also includes nucleodeside/non-nucleoside polymers. The term also includes nucleoside/non-nucleoside polymers wherein one or more of the phosphorus group linkage between monomeric units has been replaced by a non-phosphorous linkage such as a formacetal linkage, a thioformacetal linkage, a morpholino linkage, a sulfamate linkage, a silyl linkage, a carbamate linkage, an amide linkage, a guanidine linkage, a nitroxide linkage or a substituted hydrazine linkage. It also includes nucleoside/non-nucleoside polymers wherein both the sugar and the carbon moiety have been replaced or modified such as morpholino base analogs, or polyamide base analogs. It also includes nucleoside/non-nucleoside polymers wherein the base, the sugar, and the phosphate backbone of the non-nucleoside are either replaced by a non-nucleoside moiety or wherein a non-nucleoside moiety is inserted into the nucleoside/non-nucleoside polymer. Optionally, said non-nucleoside moiety may serve to link other small molecules which may interact with target sequences or alter uptake into target cells.
Lipid Aggregate is a term that includes liposomes of all types both unilamellar and multilamellar as well as micelles and more amorphous aggregates of cationic lipid or lipid mixed with amphipathic lipids such as phospholipids.
Target Cell refers to any cell to which a desired compound is delivered, using a lipid aggregate as carrier for the desired compound.
Transfection is used herein to mean the delivery of expressible nucleic acid to a target cell, such that the target cell is rendered capable of expressing said nucleic acid. It will be understood that the term xe2x80x9cnucleic acidxe2x80x9d includes both DNA and RNA without regard, to molecular weight, and the term xe2x80x9cexpressionxe2x80x9d means any manifestation of the functional presence of the nucleic acid within the cell, including without limitation, both transient expression and stable expression.
Delivery is used to denote a process by which a desired compound is transferred to a target cell such that the desired compound is ultimately located inside the target cell or in, or on the target cell membrane. In many uses of the compounds of the invention, the desired compound is not readily taken up by the target cell and delivery via lipid aggregates is a means for getting the desired compound into the cell. In certain uses, especially under in vivo conditions, delivery to a specific target cell type is preferable and can be facilitated by compounds of the invention.
The generic structure of functionally active cationic lipids requires three contiguous moities, e.g. cationic-head-group/linker/lipid-tail group. While a wide range of structures can be envisioned for each of the three moieties, it has been demonstrated that there is no a priori means to predict which cationic lipid will successfully transfect anionic macromolecules into a particular cell line. The property of a cationic lipid to be formulated with an anionic macromolecule which will then successfully transfect a cell line is empirical. We demonstrate the abilities of novel cationic lipids which are chemically linked into multimeric constructions to enhance the uptake of macromolecules.
The novel carbamate-based cationic lipids of the present invention have the general structure: 
comprising any salt, solvate, or enantiomers thereof. The symbols R1, R2, X, n, and m are described as follows.
R1 represents the lipid-tail group of the carbamate-based cationic lipid and may be a variety of lipophilic moieties, in particular, these include for example, a straight chain alkyl of 1 to about 24 carbon atoms, a straight chain alkenyl of 2 to about 24 carbon atoms, a symmetrical branched alkyl or alkenyl of about 10 to about 50 carbon atoms, a unsymmetrical branched alkyl or alkenyl of about 10 to about 50 carbon atoms, a steroidyl moiety, a glyceryl derivative, a amine derivative, or OCH(R4R5) wherein R4 and R5 are straight chain or branched alkyl moieties of about 10 to about 30 carbon atoms.
In the case where R1 is a steriodal moiety a variety of such moieties may be utilized including for example pregnenolone, progesterone, cortisol, corticosterone, aldosterone, androstenedione, testosterone, or cholesterol or analogs thereof. A cholesteryl moiety is particularly prefered.
In particular, R1 may be a 3-(1,2-diacyl)propane 1,2-diol moiety, a 3-(1,2-dialkyl)propane 1,2-diol moiety, a 3-O-1,2-diacylglyceryl moiety, or a 3-O-1,2-dialkylglyceryl moiety. In the case, when R1 is a 3-(1,2-diacyl)propane 1,2-diol moiety or a 3-O-1,2-diacylglyceryl moiety the diacyl group may be an alkanoic acid of about 10 to about 30 carbon atoms or an alkenoic acid of about 10 to about 30 carbon atoms. Similarly, when R1 is a 3-(1,2-dialkyl)propane 1,2-diol moiety or a 3-O-1,2-diacylglyceryl moiety the alkyl moieties may be an alkyl group of about 10 to about 30 carbon atoms or an alkenyl group of about 10 to about 30 carbon atoms. These groups may be straight chain, symmetrically, or unsymmetrically branched alkyl and alkenyl groups.
In either case, when R1 is a propanediol moiety or a glyceryl derivative comprising an alkanoic acid the acid is preferably a stearic acid. Similarly, when these derivative comprise an alkenoic acid the acid is preferably a palmitoic acid or an oleic acid,
R2 represents the cationic head group of the carbamate-based cationic lipid and may be a variety of positively charged moieties including, for example, an amino acid residue having a positively charged group on the side chain, an alkylamine moiety of about 3 to about 10 carbon atoms, a fluoroalkylamine moiety or a perfluoroalkylamine moiety of 1 to about 6 carbon atoms; an arylamine moiety or an aralkylamine moiety of about 5 to about 10 carbon atoms, a guanidinium moiety, an enamine moiety, an aromatic or non-aromatic cyclic amine moiety of about 5 to about 10 carbon atoms, an amidine moiety, an isothiourea moiety, a heterocyclic amine moiety, a heterocyclic moiety and an alkyl moiety of 1 to about 6 carbon atoms substituted with a substituent selected from the group consisting of NH2, C(xe2x95x90O)NH2, NHR6, C(xe2x95x90O)NHR6, NHR6R7 or C(xe2x95x90O)NHR6R7, wherein R6 and R7, are independently selected from an alkyl moiety of 1 to about 24 carbon atoms, an alkenyl moiety of 2 to about 24 carbon atoms, an aryl moiety of about 5 to about 20 carbon atoms, and an aralkyl moiety of about 6 to about 25 carbon atoms.
In particular when R2 is an amino acid residue it may be, for example, lysine, arginine, histidine, ornithine, or an amino acid analog. Although R2 may be a variety of positively charged amino acid analogs, specific examples include 3-carboxyspermidine, 5-carboxyspermidine, 6-carboxyspermine and a monoalkyl, dialkyl, or peralkyl substituted derivative which is substituted on one or more amine nitrogens with an alkyl group of 1 to about 6 carbon atoms.
The linker comprises the structure joining the head group, R1 to the lipid-tail group, R2. This structure includes Y which may be an oxygen or a nitrogen and a series of xe2x80x94CH2xe2x80x94 groups, the number of which, is indicated by the letter n. n is an integer ranging from 1 to 8, in particular cases it ranges from 2 to 6 and in specific instances the integer is 2 to 4.
The counterion represented by Xxe2x88x92is an anion or a polyanion that binds to the positively charged groups present on the carbamic acid-based cationic lipid via charge-charge interactions. When these cationic lipids are to be used in vivo the anion or polyanion should be pharmaceutically acceptable.
m is an integer indicating the number of anions or polyanions associated with the cationic lipid. In particular this integer ranges in magnitude from 0 to a number equivalent to the positive charge(s) present on the lipid.
The cationic lipids of the present invention include salts, solvates, or enantiomeric isomers resulting from any or all asymmetric atoms present in the lipid. Included in the scope of the invention are racemic mixtures, diastereomeric mixtures, optical isomers or synthetic optical isomers which are isolated or substantially free of their enantiomeric or diasteriomeric partners. The racemic mixtures may be separated into their individual, substantially optically pure isomers by techniques known in the art, such as, for example, the separation of diastereomeric salts formed with optically active acid or base adjuncts followed by conversion back to the optically active substances. In most instances, the desired optical isomer is synthesized by means of stereospecific reactions, beginning with the appropriate stereoisomer of the desired starting material. Methods and theories used to obtain enriched and resolved isomers have been described (Jacques et al., xe2x80x9cEnantiomers, Racemates and Resolutions.xe2x80x9d Kreiger, Malabar, Fla., 1991).
The salts include pharmaceutically or physiologically acceptable non-toxic salts of these compounds. Such salts may include those derived by combination of appropriate cations such as alkali and alkaline earth metal ions or ammonium and quaternary amino ions with the acid anion moiety of the carbamate or carbamothioate acid group present in polynucleotides. Suitable salts include for example, acid addition salts such as HCl, HBr, HF, HI, H2 SO4, and trifluoroacetate. The salts may be formed from acid addition of certain organic and inorganic acids, e.g., HCl, Hbr, H2 SO4, amino acids or organic sulfonic acids, with basic centers, (e.g. amines), or with acidic groups. The composition herein also comprise compounds of the invention in their un-ionized, as well as zwitterionic forms.
Exemplary invention cationic lipids have the structures shown in the Summary of the Invention above.
The cationic lipids form aggregates with polyanionic macromolecules such as oligonucleotides, oligomers, peptides, or polypeptides through attraction between the positively charged lipid and the negatively charged polyanionic macromolecule. The aggregates may comprise multiamellar or unilamellar liposomes or other particles. Hydrophobic interactions between the cationic lipids and the hydrophobic substituents in the polyanionic macromolecule such as aromatic and alkyl moieties may also facilitate aggregate formation. Cationic lipids have been shown to efficiently deliver nucleic acids and peptides into cells in the presence of serum and thus are suitable for use in vivo or ex vivo.
Cationic lipid-polyanionic macromolecule aggregates may be formed by a variety of method known in the art. Representative methods are disclosed by Felgner et al., supra; Eppstein et al. supra; Behr et al. supra; Bangham, A. et al. M. Mol. Biol. 23:238, 1965; Olson, F. et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, P. et al. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, E. et al. Biochim. Biophys. Acta 775:169, 1984; Kim, S. et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, M. et al. Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion. See, e.g., Mayer, L. et al. Biochim. Biophys. Acta 858:161, 1986. Microfluidization is used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, E., supra). In general aggregates may be formed by preparing lipid particles consisting of either (1) a cationic lipid of the invention or (2) a cationic lipid mixed with a colipid, followed by adding a polyanionic macromolecule to the lipid particles at about room temperature (about 18 to 26xc2x0 C.). In general, conditions are chosen that are not conducive to deprotection of protected groups. The mixture is then allowed to form an aggregate over a period of about 10 minutes to about 20 hours, with about 15 to 60 minutes most conveniently used. The complexes may be formed over a longer period, but additional enhancement of transfection efficiency will not usually be gained by a longer period of complexing. Colipids that are suitable for preparing lipid aggregates with the cationic lipids of the present invention are dimyristoylphosphatidylethanolamine, dipalmitoyl-phosphatidylethanolamine, palmitoyloleolphosphatidyl-ethanolamine, cholesterol, distearoyalphosphatidyl-ethanolamine, phosphatidylethanolamine covalently linked to polyethylene glycol and mixtures of these colipids.
The optimal cationic lipid:colipid ratios for a given cationic lipid is determined by mixing experiments to prepare lipid mixtures for aggregation with a polyanionic macromolecule using cationic lipid:colipid ratios between about 1:0 and 1:10. Methods to determine optimal cationic lipid:colipid ratios have been described (see, Felgner, infra). Each lipid mixture is optionally tested using more than one oligonucleotide-lipid mixture having different nucleic acid:lipid molar ratios to optimize the oligonucleotide:lipid ratio.
Suitable molar ratios of cationic lipid:colipid are about 0.1:1 to 1:0.1 , 0.2:1 to 1:0.2, 0.4:1 to 1:0.4, or 0.6:1 to 1:0.6. Lipid particle preparation containing increasing molar proportions of colipid have been found to enhance oligonucleotide transfection into cells with increasing colipid concentrations.
In addition, the cationic lipids can be used together in admixture, or different concentrations of two or more cationic lipids in admixture, with or without, colipid.
Liposomes or aggregates are conveniently prepared by first drying the lipids in solvent (such as chloroform) under reduced pressure. The lipids are then hydrated and converted to liposomes or aggregates by adding water or low ionic strength buffer (usually less than about 200 mM total ion concentration) followed by agitating (such as vortexing and/or sonication) and/or freeze/thaw treatments. The size of the aggregates or liposomes formed range from about 40 nm to 600 nm in diameter.
The amount of an oligonucleotide delivered to a representative cell by at least some of the cationic lipids was found to be significantly greater than the amount delivered by commercially available transfection lipids. The amount of oligonucleotide delivered into cells was estimated to be about 2- to 100-fold greater for these cationic lipids of the invention based on the observed fluorescence intensity of transfected cells after transfection using a fluorescently labeled oligonucleotide. The cationic lipids described herein also transfect some cell types that are not detectably transfected by commercial lipids. Functionality of cationic lipid-DNA aggregates was demonstrated by assaying for the gene product of the exogenous DNA. Similarly the functionality of cationic lipid-oligonucleotide aggregates were demonstrated by antisense inhibition of a gene product.
The cationic lipids described herein also differed from commercially available lipids by efficiently delivering an oligonucleotide into cells in tissue culture over a range of cell confluency from about 50 to 100%. Most commercially available lipids require cells that are at a relatively narrow confluency range for optimal transfection efficiency. For example, Lipofectin(trademark) requires cells that are 70-80% confluent for transfecting the highest proportion of cells in a population. The cationic lipids described herein may be used to transfect cells that are about 10-50% confluent, but toxicity of the lipids was more pronounced, relative to that seen using cells that are about 50-100% confluent. In general, the cationic lipids transfected cells that were about 60-100% confluent with minimal toxicity and optimal efficiency. Confluency ranges of 60-95% or 60-90% are thus convenient for transfection protocols with most cell lines in tissue culture.
The cationic lipid aggregates were used to transfect cells in tissue culture and the RNA and the DNA encoded gene products were expressed in the transfected cells.
The cationic lipid aggregates may be formed with a variety of macromolecules such as oligonucleotides and oligomers. Oligonucleotides used in aggregate formation may be single stranded or double stranded DNA or RNA, oligonucleotide analogs, and plasmids.
In general, relatively large oligonucleotides such as plasmids or mRNAs will carry one or more genes that are to be expressed in a transfected cell, while comparatively small oligonucleotides will comprise (1) a base sequence that is complementary (via Watson Crick or Hoogsteen binding) to a DNA or RNA sequence present in the cell or (2) a base sequence that permits oligonucleotide binding to a molecule inside a cell such as a peptide, protein or glycoprotein. Exemplary RNAs include ribozymes and antisense RNA sequences that are complementary to a target RNA sequence in a cell
An Oligonucleotide may be a single stranded unmodified DNA or RNA comprising (a) the purine or pyrimidine bases guanine, adenine, cytosine, thymine and/or uracil: (b) ribose or deoxyribose; and (c) a phosphodiester group that linkage adjacent nucleoside moieties. Oligonucleotides typically comprise 2 to about 100 linked nucleosides. Typical oligonucleotides range in size from 2-10, 2-15, 2-20, 2-25, 2-30, 2-50, 8-20, 8-30 or 2-100 linked nucleotides. Oligonucleotides are usually linear with uniform polarity and, when regions of inverted polarity are present, such regions comprise no more than one polarity inversion per 10 nucleotides. One inversion per 20 nucleotides is typical. Oligonucleotides can also be circular, branched or double-stranded. Antisense oligonucleotides generally will comprise a sequence of about from 8-30 bases or about 8-50 bases that is substantially complementary to a DNA or RNA base sequence present in the cell. The size of oligonucleotide that is delivered into a cell is limited only by the size of polyanionic macromolecules that can reasonably be prepared and thus DNA or RNA that is 0.1 to 1 Kilobase (Kb), 1 to 20 Kb, 20 Kb to 40 Kb or 40 Kb to 1,000 Kb in length may be delivered into cells.
Oligonucleotides also include DNA or RNA comprising one or More covalent modifications. Covalent modifications include (a) substitution of an oxygen atom in the phosphodiester linkage of an polynucleotide with a sulfur atom, a methyl group or the like, (b) replacement of the phosphodiester group with a nonphosphorus moiety such as xe2x80x94Oxe2x80x94CH2Oxe2x80x94, Sxe2x80x94CH2Oxe2x80x94 or O CH2Oxe2x80x94S, and (c) replacement of the phosphodiester group with a phosphate analog such as xe2x80x94Oxe2x80x94P(S)(O)xe2x80x94O, xe2x80x94Oxe2x80x94P(S)(S)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94P(CH3)(O)xe2x80x94O or xe2x80x94Oxe2x80x94P(NHR10)(O)xe2x80x94Oxe2x80x94 where R10 is alkyl (C1-6), or an alkyl ether (C1-6). Such substitutions may constitute from about 10% to 100% or about 20 to about 80% of the phosphodiester groups in unmodified DNA or RNA. Other modifications include substitutions of or on sugar moiety such as morpholino, arabinose 2xe2x80x2-fluororibose, 2xe2x80x2-fluoroarabinose, 2xe2x80x2xe2x80x94Oxe2x80x94methylribose or 2xe2x80x2-O-allylribose. Oligonucleotides and methods to synthesize them have been described (for example see: U.S. patent application Ser. Nos. 08/154,013, filed Nov. 16, 1993, 08/154,014, filed Nov. 16, 1993, 08/001,179, filed Jun. 6, 1993, and 08/233,778 filed May 4, 1994, PCT/US90/03138, PCT/US90/06128, PCT/US90/06090, PCT/US90/06110, PCT/US92/03385, PCT/US91/08811, PCT/US91/03680, PCT/US91/06855, PCT/US91/01141, PCT/US92/10115, PCT/US92/10793, PCT/US93/05110, PCT/US93/05202, PCT/US92/04294, WO 86/05518, WO 89/12060, WO 91/08213, WO 90/15065, WO 91/15500, WO 92/02258, WO 92/20702, WO 92/20822, WO 92/20823, U.S. Pat. No.: 5,214,136 and Uhlmann Chem Rev. 90:543, 1990). Oligonucleotides are usually linear with uniform polarity and, when regions of inverted polarity are present, such regions comprise. no more than one polarity inversion per 10 nucleotides, One inversion per 20 nucleotides is typical. Oligonucleotides may also be circular, branched or double-stranded.
The linkage between the nucleotides of the oligonucleotide may be a variety of moieties including both phosphorus-containing moieties and non phosphorus-containing moieties such as formacetal, thioformacetal, riboacetal and the like. A linkage usually comprises 2 or 3 atoms between the 5xe2x80x2 position of a nucleotide and the 2xe2x80x2 or 3xe2x80x2 position of an adjacent nucleotide. However, other synthetic linkers may contain greater than 3 atoms.
The bases contained in the oligonucleotide may be unmodified or modified or natural or unatural purine or pyrimidine bases and may be in the xcex1 or xcex2 anomer. Such bases may be selected to enhance the affinity of oligonucleotide binding to its complementary sequence relative to bases found in native DNA or RNA. However, it is preferable that modified bases are not incorporated into an oligonucleotide to an extent that it is unable to bind to complementary sequences to produce a detectably stable duplex or triplex.
Exemplary bases include adenine, cytosine, guanine, hypoxanthine, inosine, thymine, uracil, xanthine, 2-aminopurine, 2,6-diaminopurine, 5-(4-methylthiazol-2-yl)uracil, 5-(5-methylthiazol-2-yl)uracil, 5-(4-methylthiazol-2-yl)cytosine, 5-(5-methylthiazol-2-yl)cytosine and the like. Other exemplary bases include alkylated or alkynylated bases having substitutions at, for example, the 5 position of pyrimidines that results in a pyrimidine base other than uracil, thymine or cytosine, (i.e., 5-methylcytosine, 5-(1-propynyl)cytosine, 5-(1-butynyl)cytosine, 5-(1-butynyl)uracil, 5-(1-propynyl)uracil and the like). The use of modified bases or base analogs in oligonucleotides have been previously described (see PCT/US92/10115; PCT/US91/08811; PCT/US92/09195; WO 92/09705; WO 92/02258; Nikiforov, et al., Tet. Lett. 33:2379, 1992; Clivio, et al., Tet. Lett. 33:65, 1992; Nikiforov, et al., Tet. Lett. 32:2505, 1991; Xu, et al., Tet. Lett. 32:2817, 1991; Clivio, et al., Tet. Lett. 33:69, 1992; Connolly, et al., Nucl. Acids Res. 17:4957, 1989).
Aggregates may comprise oligonucleotides or oligomers encoding a therapeutic or diagnostic polypeptide. Examples of such polypeptides include histocompatibility antigens, cell adhesion molecules, cytokines, antibodies, antibody fragments, cell receptor subunits, cell receptors, intracellular enzymes and extracellular enzymes or a fragment of any of these. The oligonucleotides also may optionally comprise expression control sequences and generally will comprise a transcriptional unit comprising a transcriptional promoter, an enhancer, a transcriptional terminator, an operator or other expression control sequences.
Oligonucleotides used to form aggregates for transfecting a cell may be present as more than one expression vector. Thus, 1, 2, or 3 or more different expression vectors may be delivered into a cell as desired. Expression vectors will typically express 1, 2, or 3 genes when transfected into a cell, although many genes may be present such as when a herpes virus vector or a yeast artificial chromosome is delivered into a cell. Expression vectors that are introduced into a cell can encode selectable markers (e.g. neomycin phosphotransferase, thymidine kinase, xanthine-guanine phosphoribosyl-transferase, and the like) or biologically active proteins such as metabolic enzymes or functional proteins (e.g. immunoglobulin genes, cell receptor genes, cytokines (e.g. IL-2, IL-4, GM-CSF, xcex3-INF and the like), or genes that encode enzymes that mediate purine or pyrimidine metabolism and the like).
The nucleic acid sequence of the oligonulcleotide coding for specific genes of interest may be retrieved, without undue experimentation, from the GenBank of EMBL DNA libraries. Such sequences may include coding sequences, for example, the coding sequences for structural proteins, hormones, receptors and the like, and the DNA sequences for other DNAs of interest, for example, transcriptional and translational regulatory elements (promoters, enhancers, terminators, signal sequences and the like), vectors (integrating or autonomous) and the like. Non-limiting examples of DNA sequences which may be introduced into cells with the reagent of the invention include those sequences coding for fibroblast growth factor (see WO 87/01728); ciliary neurotrophic factor (Lin et al., Science, 246:1023, 1989; human interferon-xcex1 receptor (Uze, et al., Cell, 60:225,,1990; the interleukins and their receptors (reviewed in Mizal, FASEB J., 3:2379, 1989; hybrid interferons (see EPO 051,873); the RNA genome of human rhinovirus (Callahan, Proc. Natl. Acad. Sci., 82:732, 1985; antibodies including chimeric antibodies (see U.S. Pat. No.: 4,816,567); reverse transcriptase (see Moelling, et al., J. Virol., 32:370, 1979; human CD4 and soluble forms thereof (Maddon et al., Cell, 47:333, 1986, see WO 88/01304 and WO 89/01940); and EPO 330,191, which discloses a rapid immunoselection cloning method useful for the cloning of a large number of desired proteins.
Aggregates can be used in antisense inhibition of gene expression in a cell by delivering an antisense oligonucleotide into the cell (see Wagner, Science 260:1510, 1993 and WO 93/10820). Such oligonucleotides will generally comprise a base sequence that is complementary to a target RNA sequence that is expressed by the cell. However, the oligonucleotide may regulate intracellular gene expression by binding to an intracellular nucleic acid binding protein (see Clusel, Nucl. Acids Res. 21:3405, 1993) or by binding to an intracellular protein or organelle that is not known to bind to nucleic acids (see WO 92/14843). A cell that is blocked for expression of a specific gene(s) is useful for manufacturing and therapeutic applications. Exemplary manufacturing uses include inhibiting protease synthesis in a cell to increase production of a protein for a therapeutic or diagnostic application (e.g., reduce target protein degradation caused by the protease). Exemplary therapeutic applications include inhibiting synthesis of cell surface antigens to reduce rejection and/or to induce immunologic tolerance of the cell either after it is implanted into a subject or when the cell is transfected in vivo (e.g. histocompatibility antigens, such as MHC class II genes, and the like).
Methods to introduce aggregates into cells in vitro and in vivo have been previously described (see U.S. Pat. Nos.: 5,283,185 and 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol Chem 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.
Entry of liposomes or aggregates into cells may be by endocytosis or by fusion of the liposome or aggregate with the cell membrane. When fusion takes place, the liposomal membrane is integrated into the cell membrane and the aqueous contents of the liposome merge with the fluid in the cell.
Endocytosis of liposomes occurs in a limited class of cells; those that are phagocytic, or able to ingest foreign particles. When phagocytic cells take up liposomes or aggregates, the cells move the spheres into subcellular organelles known as lysosomes, where the liposomal membranes are thought to be degraded. From the lysosome, the liposomal lipid components probably migrate outward to become part of cell""s membranes and other liposomal components that resist lysosomal degradation (such as modified oligonucleotides or oligomers) may enter the cytoplasm.
Lipid fusion involves the transfer of individual lipid molecules from the liposome or aggregate into the plasma membrane (and vice versa); the aqueous contents of the liposome may then enter the cell. For lipid exchange to take place, the liposomal lipid must have a particular chemistry in relation to the target call. Once a liposomal lipid joins the cell membrane it can either remain in the membrane for a period of time or be redistributed to a variety of intracellular membranes. The invention lipids can be used to deliver an expression vector into a cell for manufacturing or therapeutic use. The expression vectors can be used in gene therapy protocols to deliver a therapeutically useful protein to a cell or for delivering nucleic acids encoding molecules that encode therapeutically useful proteins or proteins that can generate an immune response in a host for vaccine or other immunomodulatory purposes according to known methods (see U.S. Pat. Nos.: 5,399,346 and 5,336,615, WO 94/21807 and WO 94/12629). The vector-transformed cell can be used to produce commercially useful cell lines, such as a cell line for producing therapeutic proteins or enzymes (e.g. erythropoietin, and the like), growth factors (e.g. human growth hormone, and the like) or other proteins. The aggregates may be utilized to develop cell lines for gene therapy applications in humans or other species including murine, feline, bovine, equine, ovine or non-human primate species. The aggregates may be used in the presence of serum and will thus deliver polyanionic macromolecules into cells in tissue culture medium containing serum in vitro or in an animal in vivo.
The following examples are offered by way of illustration and not by way of limitation.